Phytopathogenic fungi are organisms responsible for several plant diseases in different crops around the world, causing very important economic losses to the farmers. Fungi have a complicated life cycle, normally with asexual and sexual reproduction that involves the formation of different reproductive structures. Moreover, during plant disease, fungi produce different components that are essential to complete the infection process (enzymes, toxins, etc) and are named “pathogenicity factors”. Two dimensional gel electrophoresis (2-DE) has been widely used to study the proteome of a good number of microorganisms. However, few of them have been carried out to study the proteome of phytopathogenic fungi, mainly due to the difficulty of obtaining fungal protein extracts and/or the lack of available fungal protein databases. This review shows the strategies that can be addressed to overcome those problems and how different approaches have resulted to be useful in (i) obtaining the proteomic maps of several phytopathogenic fungi, (ii) determining fungal proteins involved in the formation of infective structures and (iii) studying the production of extracellular proteins or pathogenicity factors in phytopathogenic fungi. Fungal proteomic is still at an early stage, but recent reports show that it is a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research.